Application Note

Using Advanced Polymer Chromatography with Quaternary Solvent Manager and Evaporative Light Scattering Detection for Gradient Polymer Elution Chromatography

Jennifer Gough, Will Martin, Neil J. Lander

Waters Corporation

Abstract

The APC with QSM is specifically designed for aggressive solvents used in the polymer

industry. The Waters ACQUITY Advanced Polymer Chromatography (APC) System is designed to

operate consistently using harsh solvents like THF, chloroform, and dimethyl sulfoxide. By adding

the flexibility of a quaternary pump to the industry-accepted APC system, polymer analysis

techniques are no longer limited to HPLC technology.

Benefits

p-QSM with aggressive solvent compatibility right out of the box.■

Reverse-phase, normal-phase, and size-based separations provide flexible analysis.■

The low dispersion APC with p-QSM uses less solvent than HPLC or GPC, which makes it more

sustainable

■

High resolution with sub-3-μm BEH column technology■

Introduction

The gradient polymer elution chromatography (GPEC) chromatogram in Figure 1 has a run time of

30 minutes using an Alliance HPLC (high-performance liquid chromatography) system. If this

method is transferred to an UltraPerformance Liquid Chromatography (UPLC) system, the run time

could be significantly shortened. However, most UPLC systems are not compatible with organic

solvents, such as tetrahydrofuran (THF), and are not robust enough to constantly use 100% organic

solvent.

The Waters ACQUITY Advanced Polymer Chromatography (APC) System is designed to operate

consistently using harsh solvents like THF, chloroform, and dimethyl sulfoxide. Originally, the APC

system was designed for gel permeation chromatography (GPC) using an isocratic pump. By adding

the polymer quaternary solvent manager (p-QSM) for gradient elution and an evaporative light

scattering detector (ELSD) to the APC system, GPEC can be accomplished in less than 10 minutes

(Figure 1).

Figure 1. GPEC comparison of HPLC and UPLC analysis times.

Experimental

System: APC with p-QSM

Column: XBridge BEH C8, XP, 2.5 μ

m, 3.0 × 100 mm (p/n

186006047)

Mobile phase A/B: Methanol

Mobile phase C/D: Tetrahydrofuran

Flow rate: 0.7 mL/min

Column temp.: 40 °C

Injection volume: 1 μL

Sample concentration: PMMA (1 mg/mL), PS

(0.12 mg/mL), PBD (0.5

mg/mL)

Gradient:

Sr.

No

Time Flow

(mL/min)

%A %B %C %D Curve

1 Initial 0.700 100.0 0.0 0.0 0.0 Initial

2 1.00 0.700 60.0 0.0 40.0 0.0 11

3 2.00 0.700 35.0 0.0 65.0 0.0 11

4 3.00 0.700 15.0 0.0 85.0 0.0 11

5 4.00 0.700 15.0 0.0 85.0 0.0 11

6 5.00 0.700 0.0 0.0 100.0 0.0 6

7 6.00 0.700 100.0 0.0 0.0 0.0 6

8 7.00 0.700 100.0 0.0 0.0 0.0 11

In order to precipitate a polymer onto the column, the polymer solubility in the gradient blend of

solvents needs to be determined. The accepted method in literature is to titrate a known

concentration of polymer solution (10–2 to 10–3 mg/mL) with a non-solvent (poor solvent). As the

titration progresses, the polymer solution will appear cloudy as the polymer aggregates and

precipitates: the polymer is repelled by the non-solvent and the polymer molecules are forced

together in aggregation. The volume fraction of the non-solvent is calculated using Equation 1,

where phi (F) equals the cloud point (CP) divided by 100, which equals the volume of the non-

solvent (Vns) divided by the sum of polymer volume, good solvent volume, and non-solvent volume

(Vp + Vs + Vns). This technique is called cloud-point determination.4,5

Eq 1. F = CP/100 = Vns / Vp + Vs + VNS

If the linear gradient cannot be optimized to achieve reproducible peak areas, a step gradient may

be needed. The GPEC of three polymers, polystyrene (PS), polybutadiene (PBD), and

polymethylmethacrylate (PMMA), are used for this experiment (Table 1). The linear gradient

produces poor peak area reproducibility for PS and PMMA, and the PBD does not elute.

Table 1. Polymers used in GPEC experiment.

Therefore, a step gradient method is used to control the amount of time the polymers spend in the

insoluble regions of the gradient. A steep curve is used for the insoluble regions (11) of the gradient

profile, while the soluble region is a less steep curve (6) as seen in the gradient table and resulting

chromatography (Figure 2).

Figure 2. Empower overlay of the GPEC analysis of PMMA, PS, and PBD.

By using the Empower 3 CDS custom calculations and report features, there is no need to export

data into a separate spreadsheet. The report in Figure 3 has the peak area ratios and the gradient

ratios calculated within Empower. The complete GPEC project and detailed GPEC guide are

available on the Waters website and in the Empower marketplace.

Figure 3. Empower report for the GPEC analysis of three separate

injections with gradient custom calculation.

Results and Discussion

GPEC is not GPC. In the example chromatograms of Figure 4, three chemically different polymers

co-elute using a GPC separation, because they have similar hydrodynamic volumes. If reversed-

phase separation is used to separate these polymers, they would remain partially soluble throughout

the separation and not baseline separate: partially overlapping. The GPEC method purposefully

precipitates the polymers temporarily onto the column or column frit until the gradient ratio of good

solvent reaches the polymer’s solubility point, releasing the polymer from the surface of the column.

Instead of using the size-based separation of GPC, the polymers are separated by their chemical

solubility using GPEC. GPEC is an LC application and is completely different from a GPC

application.1

Figure 4. GPC and GPEC of three polymers using an Alliance System.

The APC system designed for GPEC has a new configuration (Figure 5). The polymer isocratic

solvent manager (p-ISM) has been replaced with a polymer quaternary solvent manager (p-QSM) to

accommodate gradient elution with 100% organic solvent. The refractive index (RI) detector has

been replaced with an evaporative light scattering detector (ELSD), because RI is not compatible

with gradient elution. A photodiode array (PDA) is not used for this application example, because

not all polymers are UV active.2,3

Figure 5. APC System with p-QSM

and ELSD.

Conclusion

GPEC is a complementary analysis to GPC, when co-eluting peaks interfere with determining

polymer ratios in a mixture. This uncommon analysis technique has been used for decades but has

been limited to traditional HPLC instruments. UPLC instruments have solvent compatibility

limitations that can limit analysis options like GPEC. The APC with QSM is specifically designed for

aggressive solvents used in the polymer industry. By adding the flexibility of a quaternary pump to

the industry-accepted APC system, polymer analysis techniques are no longer limited to HPLC

technology.

References

Uliyanchenko, E.; van der Walac, S.; Schoenmakers, P.J., Challenges in Polymer Analysis by

Liquid Chromatography. Polym. Chem. 2012, 3, 2313–2335.

https://pubs.rsc.org/en/content/articlelanding/2012/py/c2py20274c#!divAbstrac.

1.

Waters ACQUITY UPLC ELS Detector, https://www.waters.com/waters/nav.htm?cid=514219.2.

ACQUITY UPLC Evaporative Light Scattering Detector Getting Started Guide,

https://www.waters.com/webassets/cms/support/docs/71500109303rd.pdf.

3.

Klumperman, B.; Cools, P.; Philipsen, H.; Staal, W. (1996), A Qualitative Study to the Influence of

Molar Mass on Retention in Gradient Polymer Elution Chromatography (GPEC). Macromol.

Symp., 110: 1–13. doi:10.1002/masy.19961100102,

https://onlinelibrary.wiley.com/doi/abs/10.1002/masy.19961100102.

4.

Klenin, V. J.; Shmakov, S.L. Features of Phase Separation in Polymeric Systems: Cloud-Point

Curves (discussion). Universal Journal of Materials Science 2013, 1 (2), 39–45.

https://pdfs.semanticscholar.org/44cf/8e3fc4396d2d8bc6bc55 b5be0b2648e70619.pdf.

5.

Featured Products

ACQUITY Advanced Polymer Chromatography System

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